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, 135 (5), 894-906

Chromosome Congression by Kinesin-5 Motor-Mediated Disassembly of Longer Kinetochore Microtubules

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Chromosome Congression by Kinesin-5 Motor-Mediated Disassembly of Longer Kinetochore Microtubules

Melissa K Gardner et al. Cell.

Abstract

During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression.

Figures

Figure 1
Figure 1. Cin8p organizes metaphase yeast kinetochores in a manner consistent with length-dependent suppression of net kMT plus-end assembly
(A) A spatial gradient in net kMT plus-end assembly mediates kinetochore congression in yeast. Kinetochores (cyan) congress to attractor zones (yellow arrows) on either side of the spindle equator (dotted line) during yeast metaphase via the plus-end assembly dynamics of kMTs (black). The kMT plus-end assembly dynamics are spatially regulated such that plus-end assembly is favored near the poles (grey) when kMTs are relatively short (favorable assembly zone shown as green gradient), and suppressed near the spindle equator (dotted line) when kMTs are relatively long (assembly suppression zone shown as red gradient). (B) A computational model of yeast metaphase kMT plus end dynamics predicts that deletion of the length-dependent promoter of kMT disassembly will result in longer kMTs and disorganized kinetochores. Conversely, overexpression will result in shorter kMTs and focused kinetochore clusters. All model parameters as in Table S2. (C) CIN8 deletion results in kinetochore disorganization and the net shifting of kinetochores towards the spindle equator, suggesting that kMTs are on average longer than they are in wild-type cells (red, Spc29-CFP pole marker; green, Cse4-GFP kinetochore marker) (scale bar 1000 nm, error bars, s.e.m.) (D) Cin8p overexpression results in clusters of kinetochores near the SPBs, suggesting that kMTs are shorter than in wild-type cells.
Figure 2
Figure 2. Cin8p promotes kMT disassembly
(A) CIN8 deletion results in flattening of the GFP-tubulin fluorescence distribution and shifting of fluorescence towards the spindle equator, suggesting that kMT lengths are increased in the mutant. (Red, Spc29 CFP SPB marker; Green, GFP-Tub1 MT marker) (scale bar 1000 nm, error bars, s.e.m.) All model parameters as in Table S2. (B) Cryo-electron tomography reveals increased mean MT length and number in cin8Δ spindles (n=5 spindles, mean spindle length =1387 nm), relative to wild-type spindles (n=4 spindles, mean spindle length=1265 nm). (C) Similar to the CIN8 deletion mutant, the motor domain mutant, cin8-F467A, which has reduced affinity for MTs, results in flattening of the GFP-tubulin fluorescence distribution and shifting of fluorescence towards the spindle equator. (D) CIN8 deletion eliminates the characteristic gradient in GFP-tubulin FRAP recovery half-time, consistent with disruption of the gradient in net kMT assembly. (E) In contrast to the cin8Δ mutants, deletion of the kinesin-8 depolymerase KIP3 does not significantly perturb kinetochore microtubule (kMT) organization.
Figure 3
Figure 3. Cin8p promotes astral MT disassembly
(A) Astral MTs (aMTs) extend from the SPBs into the cytoplasm, and are fewer in number than spindle microtubules. (B) aMT lengths were measured via GFP-tubulin fluorescence (white arrows point to plus ends; red, Spc29-CFP pole marker; green, GFP-tubulin) (scale bar 500 nm). (C) aMT lengths are increased in cin8Δ cells as compared to wild-type spindles. Cytoplasmic overexpression of Cin8p results in shorter aMTs, whether overexpression is global, as in GAL1-CIN8 overexpression experiments, or if overexpression is local in the cytoplasm, as in experiments with mutant Cin8p lacking the nuclear localization signal (cin8-nlsΔ). Both kip3Δ and cin8-F467A mutants have increased aMT lengths relative to wild-type cells, similar to cin8Δ cells.
Figure 4
Figure 4. Cin8p accumulates on kMTs in a length-dependent manner and frequently interacts with kMT plus ends
(A) In simulation, kinesin-5 motors crosslink both anti-parallel-oriented microtubules (left, magenta) and parallel-oriented microtubules (right, green). Simulated motors crosslinking parallel-oriented microtubules move to and frequently interact with kMT plus-ends. (B) Cin8-3XGFP motor movement can be observed in the spindle (horizontal scale bar, 1000 nm; vertical scale bar, 20 sec). The three arrows (yellow, cyan, and magenta) indicate three time points in the movement of a Cin8-3XGFP fluorescent spot that moves in the plus end direction. (C) Cin8p motors concentrate near kinetochores both experimentally and in simulation (Red, Ndc80-Cherry kinetochore marker; green, Cin8-GFP) (scale bar 500 nm, error bars, s.e.m.). (D) Experimentally and in simulation, Cin8-GFP fluorescence normalized to the number of tubulin polymer binding sites increases for longer kMTs. (E) Cin8-GFP FRAP half-time gradient.
Figure 5
Figure 5. Relative Distribution of Cin8-GFP and Kip3-GFP
(A) The experimental and simulated distribution of Cin8-GFP and Kip3-GFP. (B) In simulations, the crosslinking properties of Cin8p frustrates its processivity toward the plus-ends of interpolar (iMT) plus-ends, such that Cin8p visits to iMT plus-ends are rare relative to kMT plus-end visits.
Figure 6
Figure 6. Cin8p walks processively towards MT plus ends, and its distribution on aMTs mirrors its distribution on kMTs
(A) Cin8-NLSΔ-3XGFP (left, green) moves in the plus end direction on aMTs, and frequently interacts with aMT plus-ends. The three arrows (yellow, cyan, and magenta) indicate three time points in the movement of a Cin8-GFP fluorescent spot that moves in the plus end direction (red, tubulin-cherry)) (horizontal scale bar, 1500 nm; vertical scale bar, 50 sec) (B) Cin8-NLSΔ-3XGFP (green) on aMTs (tubulin-cherry, red). (C) Cin8-NLSΔ-GFP concentrates near the plus-ends of longer aMTs. (D) Experimentally and in simulation, Cin8-GFP fluorescence normalized to the number of tubulin polymer binding sites increases for longer aMTs.
Figure 7
Figure 7. A “self-organized” model for Cin8p motor mediated spindle organization
(A) A model for interaction of Cin8p motors with kMT plus-ends: Cin8p motors that crosslink parallel microtubules are plus-end directed (top). Cin8p concentrates on longer kMT plus-ends to directly promote kMT disassembly (middle). kMT depolymerization then promotes motor detachment (bottom). (B) Starting with a random distribution of kinetochores and motors in the spindle (top), motor-mediated promotion of kMT plus-end disassembly can organize the spindle into a typical metaphase bi-lobed kinetochore configuration as motors concentrate in a length-dependent fashion onto kMT plus-ends (bottom and Supplemental Movie 5). (C) Simulated images of Cin8-GFP (green) and Ndc80-cherry (red). At simulation start (t=0), motor and kinetochore fluorescence is randomly distributed in the spindle (left). After t=135 sec, simulated fluorescence distributions qualitatively reproduce experimental results (top right, simulated image; bottom, quantitative comparison to experimental data). Model parameters Table S7. (D) The self-organized model results in a gradient of catastrophe frequency (red triangles) that is similar to the theoretical catastrophe gradient depicted in Fig. 1B (red line).

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